Latest revision as of 23:46, 19 October 2016

FimH site directed mutated with RPMrel

Usage and Biology

Some substrains of E.Coli have a structure called Type 1 pili which is expressed from the Fim gene system. At the end of the pili structure there is a protein called “FimH” which is the structure that allows them to bind to the mannose sugar that is found on the surfaces of eukaryotic cells. (Sauer et al., 2016).However, the substrain that was used in this project was BL21, a non-pathogenic laboratory strain. Deleted mannose binding and replacing it with RPMrel would provide tumor specific binding (Kelly et al., 2003).The CPIEDRPMC (RPMrel) peptide can bind to five colon cancer cell lines: HT29, CaCo-2, RKO, SW480, and DLD-1. Here on we have choosen CaCo-2 that is studied commonly in METU as our candidate to show targeted thearpy.

3D Structure of FimH

Figure1. 3D Structure of FimH together with RPMrel can be seen below as Harvard BioDesign 2015 submitted in part registry.

Figure2. Our simulations were designed by the NAMD program, and CHARMM force field was applied. This CHARMM technique was used to calculate the energy usage in the simulations. The FimH and HETA models were used in the simulation were taken from an cristal model. After 100 ns, the protein could not disassociate from the ligand. We can understand that there is a strong interaction between the protein and the ligand.

DNA Gel Analysis

Figure 3:In the first 4 lanes the the gene is shown approximately 2100 base pairs and it is uncut. In the firts 4 lanes after the second ladder the gene is shown approximately 3300 base pairs and it is single cut with EcoRI. With the single cut it’s been shown that the insert is successfully in our vector.

Confirmation: PCR

Figure 4: The primers that we have designed bind and multiply the site when the insert and vector are ligated properly, forward binds to a region in vector and reverse binds to a region in insert and give a product at 781 bp for 1,2,3,4 which is K2052014.

Primers: forward 5’-CGAAAAGCCAAAACCTGG-3’-18 bp reverse 5’-GAAGCCTGCATAACGCGG-3’ -18 bp

However , single digestion and conformational PCR has been made to conform that the vector and the insert unites properly. As it can be seen in the first four lanes,PCR gave a hit at 781 bp. It has been conformed that they united well.

Charcterization

Figure 5: The images above represent our co-culture results that we have treated E. coli K12 and E. coli BL21 with CaCo2 cancer cell lines. The pictures show that the cells are sustainable for 6 hours, which is enough for our project. Our observation showed that optimal time for co-culture in our conditions is 6 hours. Then we optimized 1 hour is enough for binding and our treatment trials going to be performed in 5 hours. We expected to see increase in the cell death in a time dependent manner. According to our observation our BL21 forms clusters. However, Harvard BioDesign used E. coli K12 and it was challenging to see them without staining. therefore, BL21 was the best candidate in our case. The protocoles are explained below.

E.Coli BL21 (DE3) & E.Coli K12 MG 1665 Coculture with CaCo-2 Trial Protocol

E.Coli cultures were grown overnight and used in the early stationary phase.

Started from an overnight pre-cultured , stationary phase ( OD > 2) 100 μL sample ( E.Coli : pre-culture ) +10mL LB 37°C , 200 rpm , 16 hours OD600 measurement → OD600 = 1.0 means we have 8 x 10^8 cells 1mL , OD600 = 2.0 → 1,6 x 10^9 cells 1mL ( assumed as stationary phase ) Coculture : CaCo-2 cells were infected with a bacteria at a multiplicity of infection (MOI) of 1000 in 10 mL of cell culture media lacking antibiotics.

This means for 1 cell- 1000 bacteria.

We are growing 3 x 10^4 cell / well, therefore 3 x 10^7 bacteria is required. Overall, 1x 10^9 from each strain is needed.

Determine the amount of well for co-culture. i.e: K12- 25 well x (3 x 10^7 ) = 75 x 10^7 needed

2. Measure OD600 and find out how many cell/mL you have. i.e; We have 12,8 x 10^8 /mL in culture of bacteria.

3. Decide them to find out the amount to centrifuge. i.e; (75 x 10^7) / (12,8 x 10^8 ) =0,585≈0,600

4. Centrifuge (600 µL) from each bacterial culture.

5. Remove flow-through and resuspend in 500 µL medium. (Now, medium is used for cancer cell. However, we could create new media. (mix of EMEM&LB)

6. 500 µL media is needed for each well.Then, 500µL x 25 well= 12,5 mL

7. Prepare media (EMEM) as substracted the amount that we have used to resuspend bacterial strains. So, 12 mL + 500 µL media (resuspended bacteria) =12,5 mL

8. 3hr, 6 hr, 9hr overnight 24 well plates were prepared to carry K12, BL21 and cells only.

9. 12,5 mL of K12, BL21 and EMEM medium were distributed equally and placed into incubator. (No shaking! But maybe we need very slow shaking if we use early passage of CaCo-2.

10. After incubation, plates were washed with PBS as 500 µL for each well. (3 times)

11. 1,25 mL MTT is mixed with 11,25 PBS (total 12,5 mL) and distributed each well as 500 µL after washing.

12. After 4 hours incubation with MTT, add 500 µL SDS (as we have described preparation of SDS) to each well and leave overnight.

Co-culture and Fluoresence Microscopy

Figure 6: Upper left picture shows the cells only and next to it we have taken picture of CaCo2 cells cocultured with Gfp tagged FimH. Bottom pictures were taken under the Fluoresence Microscope but we havent seen any illumination that we have expected. We thought that the fusion of these proteins can change the folding of each protein and resulted in no accumulation of bacteria on cancer cells and any presence of GFP illumination.

After we havent seen any GFP activity we prepared a new experimental set up for Flowcytometry to be sure whether our control group(only GFP producing bacteria) is working or not and then we wanted to summarize our findings.

Flowcytometry Result

Figure 7: After measuring GFP signals we have obtained these graphs. Here upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only FimH. Middle triplet stand for only GFP expressed bacterial culture analysis which gave a peak that could be used as a control group. We have obtained the peaks in the FL1 channel of Fluorescence Microscope. The bottom triplet was GFP tagged FimH expressed bacterial group and as we have shown there is any signal depend on GFP activity.

Possible Outcomes of All Our Experimental Findings

After we did co-culture with CaCo-2 cells, we didn’t observe any clumps around cancer cells, but we observed our freely located bacteria without GFP illumination . We concluded from the results that because there wasn’t any ribosome binding site between GFP and FimH proteins, GFP tagged FimH is unfunctionally formed and the fusion affected their folding

Modeling

Molecule versus second

Figure 8: In the presence of arabinose, the figure above shows the increase in molar concentrations of TetR-LVA, Antiholin and mRNA of FimH in a time span of 10 seconds.

Molecule versus second

Figure 9: In the absence of arabinose, the figure above shows the change in molecule concentrations of Holin mRNA, Holin, Endolysin mRNA, Endolysin, Antiholin and dimer complexes in a time span of 10 seconds.

References

Krogfelt KA., Bergmans H., Klemm P. (1990, June) “Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae”

Sauer MM, Jakob RP, Eras J, Baday S, Eriş D, Navarra G, Bernèche S, Ernst B, Maier T, Glockshuber R. (2016 March 7). “Catch-bond mechanism of the bacterial adhesin FimH”

Kelly, K. A., Jones, D. A., (2003). “Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection”

Figure 1. iGEM 2015 Harvard BioDesign . Retrieved from https://parts.igem.org/Part:BBa_K1850010

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550331/

Induction protocol http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1173
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1113
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 ==Usage and Biology==
-Some substrains of E.Coli have a structure called Type 1 pili which is expressed from the Fim gene system. At the end of the pili structure there is a protein called “FimH” which is the structure that allows them to bind to the mannose sugar that is found on the surfaces of eukaryotic cells. (Sauer et al., 2016).However, the substrain that was used in this project was BL21, a non-pathogenic laboratory strain. Deleted mannose binding and replacing it with RPMrel would provide tumor specific binding (Kelly et al., 2003).The CPIEDRPMC (RPMrel) peptide can bind to five colon cancer cell lines: HT29, CaCo-2, RKO, SW480, and DLD-1. Here on we have choosen CaCo-2 that is studied commonly in METU as our candidate to show targeted thearpy.
+Some substrains of ''E.Coli'' have a structure called Type 1 pili which is expressed from the Fim gene system. At the end of the pili structure there is a protein called “FimH” which is the structure that allows them to bind to the mannose sugar that is found on the surfaces of eukaryotic cells. (Sauer et al., 2016).However, the substrain that was used in this project was BL21, a non-pathogenic laboratory strain. Deleted mannose binding and replacing it with RPMrel would provide tumor specific binding (Kelly et al., 2003).The CPIEDRPMC (RPMrel) peptide can bind to five colon cancer cell lines: HT29, CaCo-2, RKO, SW480, and DLD-1. Here on we have choosen CaCo-2 that is studied commonly in METU as our candidate to show targeted thearpy.
@@ Line 13: / Line 13: @@
 ===3D Structure of FimH===
-[[File:METU_HS_2014partinagif.gif|center|400px]]
+[[File:METU_HS_2014partinagif.gif|center|frame|100px]]
 Figure1. 3D Structure of FimH together with RPMrel can be seen below as Harvard BioDesign 2015 submitted  in part registry.
@@ Line 22: / Line 22: @@
-[[File:METUITUGf.gif|center|400px]]
+[[File:METUITUGf.gif|center|frame|400px]]
 Figure2. Our simulations were designed by the NAMD program, and CHARMM force field was  applied. This CHARMM technique was used  to calculate the energy usage in the simulations. The FimH and HETA models were  used in the simulation were  taken from an cristal model. After 100 ns, the protein could not disassociate from the ligand. We can understand that there is a strong interaction between the protein and the ligand.
@@ Line 32: / Line 32: @@
-[[File:METU HS Gel1.jpeg|900px]]
+[[File:METU HS DENEME3.jpeg|900px]]
-Figure 2.In the first 4 lanes the the gene is shown approximately 2100 base pairs and it is uncut. In the firts 4 lanes after the second ladder the gene is shown approximately 3300 base pairs and it is single cut with EcoRI. With the single cut it’s been shown that the insert  is successfully in our vector.
+Figure 3:In the first 4 lanes the the gene is shown approximately 2100 base pairs and it is uncut. In the firts 4 lanes after the second ladder the gene is shown approximately 3300 base pairs and it is single cut with EcoRI. With the single cut it’s been shown that the insert  is successfully in our vector.
-=Confirmation: PCR and Sequencing=
+=Confirmation: PCR=
+[[File:METU HS DENEMe4.jpeg|center|300px]]
+Figure 4: The primers that we have designed bind and multiply the site when the insert and vector are ligated properly, forward binds to a region in vector and reverse binds to a region in insert and give a product at 781 bp for 1,2,3,4 which is K2052014.
-[[File:METU HS Gel2.jpeg|700px]]
+Primers:
-==Figure 3==
-The primers that we have designed binds and multiply the site when the insert and vector are ligated properly, forward binds to a region in vector and reverse binds to a region in insert and give a product at 781 bp. Primers:
 forward  5’-CGAAAAGCCAAAACCTGG-3’-18 bp
 reverse  5’-GAAGCCTGCATAACGCGG-3’ -18 bp
@@ Line 56: / Line 51: @@
 =Charcterization=
-In order to characterize this part we tend to clone it into pet28a expression vector with the cutside of NcoI and SalI. Furthermore induction should be carried out as defined below ;
-===Induction Protocol===
+[[File:Metu_hs_cacolar.jpeg|center|900px]]
-If the protein is membrane-bound , expression in mutant strains C41 , BL21 DE3 and C43(DE3) could improve expression levels.
-Expression method
-Picking of a single colony from a freshly streaked plate of the expression host containing the recombinant vector. When the heterologous protein is toxic for the cells , higher expression levels are obtained by using the so-called ‘plating’ method.
-Growing of a starter culture.
-Inoculate with the picked up colony to 50 mL of LB medium containing appropriate antibiotic.
-When a larger starter culture is required , inoculate 4mL of rich media with single colony ; grow for 4-8 hours at 37°C and use this to inoculate the starter culture.
-( Don’t let cultures grow at 37°C overnight !)
-It is better to grow overnight cultures at 30°C or lower . Alternatively ,the culture can be incubated at 37°C until OD600 is approx. 1. Then store the culture at 4°C overnight. Following morning , collect the cells by centrifugation , resuspend them in fresh medium and use this to inoculate the main culture.
-Inoculation of the main culture and incubation until OD600  reaches 0,4-L. The optimal OD value depends on the culture method and the medium. For flask cultures using LB-medium on  OD600  of  0.6 recommended. To increase growth rate we carry out the cultures at 37°C until the OD for induction is reached. Then the cultures are cooled to the induction temperature in ice-water.
-note : For good ??reation , don’t use more medium then 20% of total flask volume.
-===Induction of protein Expression===
+Figure 5: The images above represent our co-culture results that we have treated E. coli K12 and  E. coli BL21 with CaCo2 cancer cell lines. The pictures show that the cells are sustainable for 6 hours, which is enough for our project. Our observation showed that optimal time for co-culture in our conditions is 6 hours. Then we optimized 1 hour is enough for binding and our treatment trials going to be performed in 5 hours. We expected to see increase in the cell death in a time dependent manner. According to our observation our BL21 forms clusters. However, Harvard BioDesign used E. coli K12 and it was challenging to see them without staining. therefore, BL21 was the best candidate in our case. The protocoles are explained below.
-Promoter
-Induction
-Typical condition
-Range
-araBAD
-Addition of L-arabinose
-.2%
-,002-0,4 %
-T7 - Iac operator
-Addition of IPTG
-mM
-,05 - 2,0 mM
-After induction the cultures are are incubated from 3 hours to overnight depending on the induction temprature. Guide lines are below ;
+===''E.Coli BL21 (DE3)'' & ''E.Coli K12 MG 1665'' Coculture with CaCo-2 Trial Protocol===
-Incubation Temperature
-Incubation Time
-°C
-Overnight
-°C
-Overnight
-°C
-Overnight
-°C
--6 hr
-°C
-hr
-Harvesting of the cell pellet by centrifugation ( 20 min at 6000 g ). Cell pellets are stored at -20°C.
+''E.Coli'' cultures were grown overnight and used in the early stationary phase.
-Cell Propagation :
-Take Medium with aspirator.
-Add 4mL PBS to wash cells.
-Add 1mL Trypsin and wait for 5 minutes.
-Take 11mL RPMI and put 6mL into a 15-falcon tube add 5mL to T-25 flask, mix well for preventing from clump formation.
-Take 4mL of cells + medium mixture and pour it into 6mL falcon tube and gently invert.
-Take 5mL of mixture and pour it into new T-25 flask.
-Cell Freezing Protocol :
-Cells were washed with 4mL PBS and Trypsin??
-After trypsinization , cells were collected with 5mL RPMI complete medium for T-25 , into two different 15mL falcons.
-Cells were centrifuged down at 100 for 5 minutes at 4°C .
-Medium aspirated and replaced with 4mL medium per falcon. ( Since we will have 8 vials at the end )
-DMSO is pour into as final concentration of 5%. (4mL x 5% = 200μL DMSO for each falcon)
-Cells were mixed gently and distributed into ??opreservation tubes , 1mL each.
-Tubes were then taken into Mr.Frosty for pre-cold treatment in -80°C.
-Tubes were left for overnight and next day taken into nitrogen tank.
-The cells were labeled according to ‘siblings number’ ?? names , sex and ?? in notebook !
-SDS preparation for MIT :
-For 10mL :
-,5mL autoclaved dH2O
-μL 0,2N HCl
-gr SDS ( add slowly and mix gently )
-EMEM Medium Preparation : 500 mL (Earl’s MEM) :
-EMEM Stock
-mL
-pen / strep
-mL
-L-glutamine ( Stock 200mM)
-mL
-NaHCO3 (7,5%)
-mL
-Na-Pyruvate (100mM)
-mL
-x Non-essential aminoacid
-mL
-FBS (20% final conc.)
-mL
-E.Coli BL21 (DE3) & E.Coli K12 MG 1665 Coculture with CaCo-2 Trial Protocol
-E.Coli cultures were grown overnight and used in the early stationary phase.
 Started from an overnight pre-cultured , stationary phase ( OD > 2)
-μL sample ( E.Coli : pre-culture ) +10mL LB
+μL sample ( ''E.Coli'' : pre-culture ) +10mL LB
 °C , 200 rpm , 16 hours
-OD600 measurement  → OD600 = 1.0 means we have  8 x 108 cells 1mL , OD600 = 2.0 → 1,6 x 109 cells 1mL ( assumed as stationary phase )
+OD600 measurement  → OD600 = 1.0 means we have  8 x 10^8 cells 1mL , OD600 = 2.0 → 1,6 x 10^9 cells 1mL ( assumed as stationary phase )
 Coculture :
 CaCo-2 cells were infected with a bacteria at a multiplicity of infection (MOI) of 1000 in 10 mL of cell culture media lacking antibiotics.
@@ Line 151: / Line 72: @@
 This means for 1 cell- 1000 bacteria.
-We are growing 3 x 104  cell / well, therefore 3 x 107  bacteria is required. Overall, 1x 109 from each strain is needed.
+We are growing 3 x 10^4  cell / well, therefore 3 x 10^7  bacteria is required. Overall, 1x 10^9 from each strain is needed.
 Determine the amount of well for co-culture.
-i.e: K12- 25 well x (3 x 107 ) = 75 x 107 needed
+i.e: K12- 25 well x (3 x 10^7 ) = 75 x 10^7 needed
 .   Measure OD600 and find out how many cell/mL you have.
-i.e; We have 12,8 x 108 /mL in culture of bacteria.
+i.e; We have 12,8 x 10^8 /mL in culture of bacteria.
 .   Decide them to find out the amount to centrifuge.
-i.e; (75 x 107) / (12,8 x 108 ) =0,585≈0,600
+i.e; (75 x 10^7) / (12,8 x 10^8 ) =0,585≈0,600
 .   Centrifuge (600 µL) from each bacterial culture.
@@ Line 170: / Line 91: @@
 .   Prepare media (EMEM) as substracted the amount that we have used to resuspend bacterial strains. So, 12 mL + 500 µL media (resuspended bacteria) =12,5 mL
+.   3hr, 6 hr, 9hr overnight 24 well plates were prepared to carry K12, BL21 and cells only.
-FIGURE 2 : Distribution of Co-Culture and control in 24 well plate
+.   12,5 mL of K12, BL21 and EMEM medium were distributed equally and placed into incubator. (No shaking! But maybe we need very slow shaking if we use early passage of  CaCo-2.
-.   3hr, 6 hr, 9hr overnight 24 well plates were prepared to carry K12, BL21 and cells only.
-.   12,5 mL of K12, BL21 and EMEM medium were distributed equally and placed into incubator. (No shaking! But maybe we need very slow shaking if we use early passage of  CaCo-2.
 .   After incubation, plates were washed with PBS as 500 µL for each well. (3 times)
@@ Line 183: / Line 102: @@
-FimH + Pet28a:
-Conformation: PCR and Sequencing
-Design Notes: Our protein coding reagon, terminator and arabinose induced promoter are digested from our whole constract with the enzymes
-==Modeling==
+===Co-culture and Fluoresence Microscopy===
+[[File:METU HS DENEME6.jpeg|center|400px]]
-[[File:Fimhgraphicformodeling.jpeg|left|700px]]
+Figure 6: Upper left picture shows the cells only and next to it we have taken picture of CaCo2 cells cocultured with Gfp tagged FimH. Bottom pictures were taken under the Fluoresence Microscope but we havent seen any illumination that we have expected. We thought that the fusion of these proteins can change the folding of each protein and resulted in no accumulation of bacteria on cancer cells and any  presence of  GFP illumination.
+*After we havent seen any GFP activity we prepared a new experimental set up for Flowcytometry to be sure whether our control group(only GFP producing bacteria) is working or not and then we wanted to summarize our findings.
+===Flowcytometry Result===
+[[File:METU_HS_DENEME66.jpeg|center|400px]]
+Figure 7: After measuring GFP signals we have obtained these graphs. Here upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only FimH. Middle triplet stand  for only GFP expressed bacterial culture analysis which gave a peak that could be used as a control group. We have obtained the peaks in the FL1 channel of Fluorescence Microscope. The bottom triplet was GFP tagged FimH expressed bacterial group and as we have shown there is any signal depend on GFP activity.
+=Possible Outcomes of All Our Experimental Findings=
+After we did co-culture with CaCo-2 cells, we didn’t observe any clumps around cancer cells, but we observed our freely located bacteria without GFP illumination . We concluded from the results that because there wasn’t any ribosome binding site between GFP and FimH proteins,  GFP tagged FimH is unfunctionally formed and the fusion affected their folding
-==Figure 4==
+==Modeling==
-In the presence of arabinose, the figure above shows the increase in molar concentrations of FimH, TetR-LVA, Antiholin and mRNA in a time span of 10 seconds.
+Molecule versus second
+[[File:Fimhgraphicformodeling.jpeg|center|900px]]
+Figure 8: In the presence of arabinose, the figure above shows the increase in molar concentrations of TetR-LVA, Antiholin and mRNA of FimH in a time span of 10 seconds.
-[[File:Killswitchgraphfimh.jpeg|left|700px]]
+Molecule versus second
+[[File:Killswitchgraphfimh.jpeg|left|700px]]
+Figure 9: In the absence of arabinose, the figure above shows the change in molecule concentrations of Holin mRNA, Holin, Endolysin mRNA, Endolysin, Antiholin and dimer complexes in a time span of 10 seconds.
@@ Line 219: / Line 151: @@
-==Figure 5==
-In the absence of arabinose, the figure above shows the change in molecule concentrations of Holin mRNA, Holin, Endolysin mRNA, Endolysin, Antiholin and dimer complexes in a time span of 10 seconds.
@@ Line 247: / Line 177: @@
 ==References==
 Krogfelt KA., Bergmans H., Klemm P. (1990, June) “Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae”

Difference between revisions of "Part:BBa K2052014"